ONCOLOGY LETTERS 8: 248-252, 2014
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A novel cyclic pentapeptide, H‑10, inhibits B16 cancer cell growth and induces cell apoptosis GENG ZHANG1, SHOUXIN LIU2, YUNJIANG LIU3, FEIFEI WANG1, JINGWEN REN1, JIFENG GU1, KAIXUAN ZHOU1 and BAOEN SHAN1 1
Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011; State Key Laboratory Breeding Base‑Hebei Province Key Laboratory of Molecular Chemistry for Drug, Hebei University of Science and Technology, Shijiazhuang, Hebei 050018; 3Breast Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
2
Received September 26, 2013; Accepted April 16, 2014 DOI: 10.3892/ol.2014.2121 Abstract. Sansalvamide A, a cyclic depsipeptide isolated from a marine fungus of the Fusarium genus, exhibits significant antitumor activity. In the present study, H‑10 (molecular formula, C38H55N5O6; molecular weight, 677.8732), a novel sansalvamide A derivative, demonstrated an inhibitory effect on the proliferation of murine melanoma B16 cells. It was confirmed that H‑10 induced the apoptosis of the B16 cells. The inhibitory rate of various concentrations of H‑10 on the B16 cells was measured by sulforhodamine B colorimetric assay, and the results revealed that the inhibitory effect of H‑10 on the B16 cells occurred in a concentration‑dependent manner. In addition, a growth curve model of the B16 cells treated with 50 µM H‑10 revealed that the effect of H‑10 also occurred in a time‑dependent manner. The apoptotic morphology of the B16 cells was observed using an optical microscope. Following the treatment of the cells with 50 µM H‑10 for 24 h, the cell apoptosis rate was analyzed using flow cytometry. The expression levels of caspase‑3, ‑8 and ‑9 were analyzed by western blot analysis, and the results indicated that H‑10 may induce the apoptosis of B16 cells. Introduction Malignant melanoma is a cancer with an increasing incidence and mortality rate. Furthermore, it is not sensitive to radiotherapy or chemotherapy, and thus presents a problem with regard to clinical treatment. Therefore, a number of studies have focused on the development of efficient and sensitive anticancer drugs. Sansalvamide A, a cyclic depsipeptide derived from a marine fungus (Fusarium), has been found to exhibit
Correspondence to: Professor Baoen Shan, Research Center, The
Fourth Hospital of Hebei Medical University, 12 Jiankang Road, Shijiazhuang, Hebei 050011, P.R. China E‑mail: [email protected]
Key words: sansalvamide A, B16, apoptosis, caspase‑3
significant antiproliferative effects in the National Cancer Institute's 60 cancer cell line panel (1,2). In recent years, the synthesis of sansalvamide A derivatives have received increasing attention. Novel sansalvamide A derivatives show improved anticancer abilities (3), suggesting that these novel compounds may prove to be valuable therapeutic agents. In the present study, a novel sansalvamide A derivative, H‑10, a cyclic pentapeptide (molecular formula, C38H55N5O6; molecular weight, 677.8732; Fig. 1), was investigated. Furthermore, this study focused on the effects of H‑10 on the growth and apotosis of rat malignant melanoma B16 cells. The results may provide a basis for future sutides of this novel compound. Materials and methods Materials. RPMI 1640, trypsin‑EDTA solution and fetal bovine serum (FBS) were purchased from Gibco‑BRL (Carlsbad, CA, USA). H‑10 cells were provided by the Hebei Province Key Laboratory of Molecular Chemistry for Drug (Shijiazhuang, China). Sulforhodamine B (SRB) was purchased from Tokyo Chemical Industry Co., Ltd (Tokoyo, Japan), and the bicinchoninic acid (BCA) kit was purchased from Shanghai Generay Biotechnology Co., Ltd. (Shanghai, China). The polyvinylidene fluoride (PVDF) membranes were purchased from Shanghai Generay Biotech Co., Ltd. The antibody against GAPDH (polyclonal rabbit anti-mouse) was purchased from Hangzhou Goodhere Biotechnology Co., Ltd. (Hangzhou, China). The antibodies against caspase-8, -9 and -3 (all polyclonal rabbit anti-mouse) were purchased from Bioworld Technology, Inc. (Minneapolis, MN, USA). The secondary fluorescence antibody (polyclonal goat anti-rabbit) was purchased from Nanjing Gene Biotech Co., Ltd. (Nanjing, China) The B16 cell line was stored at the Research Center of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). Cell culture. The cells were cultured in RPMI 1640 medium with 10% heat‑inactivated FBS and 100 µg/ml penicillin and streptomycin. The cell line was grown in 25‑cm 2 flasks in a humidified atmosphere of 5% CO2 at 37˚C, and the media were changed every second or third day. At 80‑90% confluence, the cells were digested with trypsin‑EDTA and plated in 25‑cm2
ZHANG et al: A NOVEL CYCLIC PENTAPEPTIDE, H‑10, INHIBITS B16 CELL PROLIFERATION
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Figure 1. Structure of sansalvamide A and its derivative, H‑10.
flasks with media changes every second or third day, on 24‑ or 96‑well plates.
control group was prepared simultaneously and a growth curve was generated.
Concentration‑dependent effect of H‑10 on B16 cell growth inhibition. H‑10 was dissolved in dimethyl sulfoxide (DMSO) and diluted with serum‑free medium to prepare solutions of 1,000, 500, 100, 10 and 1 µM. Single cell suspensions of B16 cells were prepared and adjusted to the indicated concentration. The cells were then inoculated in 96‑well plates (90 µl per well), with ~2,000 cells/well. Following 4 h of cell adherence, 10 µl H‑10 was added to each well to form final concentrations of 100, 50, 10, 1 and 0.1 µM. Each group was placed into three wells, while a 1% DMSO group was simultaneously prepared as the control. The SRB colorimetric method was used to calculate the percentage growth of the B16 cells treated with the various concentrations of H‑10 for 48 h.
Flow cytometric analysis of apoptotic cell death. At 80‑90% confluence, the cells were treated with 50 µM H‑10 for 24 h, while a control group was prepared. The treated and untreated cells were then harvested, washed with phosphate‑buffered saline (PBS) and fixed with 70% ethanol for 24 h. Next, the cells were centrifuged at 300 x g for 5 min and the pellet was resuspended in PBS containing 50 µg/ml propidium iodide and 10 µg/ml RNase A. The cells with 6 h. Finally, 100 µl Tris base was added and cells were agitated for 5 min. The optical density was recorded at a wavelength of 490 nm using a microplate reader. Time‑dependent effect of H‑10 on B16 cell growth inhibition. At 80‑90% confluence, the cells were harvested with trypsin, and serum‑free medium was used to produce a single‑cell suspension. The cells were then seeded in 24‑well plates at the concentration of 20,000 cells/well. After 24 h, the wells were replaced with fresh medium, including FBS. Next, the wells were treated with 50 µM H‑10 and the cell numbers were counted following 24, 48, 72, 96, 120 and 144 h. A
Detection of caspase‑8, ‑9 and ‑3 expression by western blot analysis. The cells at 80‑90% confluence were treated with 10, 30 and 50 µM H‑10 for 24 h, and a control group was prepared. The cellular protein was extracted by radioimmunoprecipitation assay lysis buffer and the concentration was measured using the BCA kit. A total of 50 µg protein was electrophoretically separated on a 10% polyacrylamide gel. The proteins were then transferred to a PVDF membrane under the conditions of 90 V and 200 mA for 60 min. Next, the membranes were incubated in antibody dilution solution (rabbit anti‑mouse caspase‑3, ‑8, ‑9 and GAPDH; 1:500) overnight at 4˚C. The blots were then incubated with the secondary fluorescence antibody (1:5,000) for 2 h in the dark, and the results were detected using the Odyssey infrared imager (LI‑COR, Inc., Lincoln, NE, USA). Statistical analysis. Statistical analysis was performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean and were analyzed by t‑test. P0.05). Following the treatment of the B16 cells with the various concentrations of H‑10 (0.1, 1, 10, 50 and 100 µM) for 48 h, the proliferation rate of the B16 cells was found to gradually decrease. Furthermore, compared with control
ONCOLOGY LETTERS 8: 248-252, 2014
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Figure 2. Effect of H‑10 on the proliferation of B16 cells measured using the sulforhodamine B colorimetric method. Compared with the control group, no significant difference was identified in the proliferation rate of the 1% DMSO group (P>0.05). H‑10 was found to cause concentration‑dependent inhibition of the B16 cells. **P
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A novel cyclic pentapeptide, H‑10, inhibits B16 cancer cell growth and induces cell apoptosis GENG ZHANG1, SHOUXIN LIU2, YUNJIANG LIU3, FEIFEI WANG1, JINGWEN REN1, JIFENG GU1, KAIXUAN ZHOU1 and BAOEN SHAN1 1
Research Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011; State Key Laboratory Breeding Base‑Hebei Province Key Laboratory of Molecular Chemistry for Drug, Hebei University of Science and Technology, Shijiazhuang, Hebei 050018; 3Breast Center, The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei 050011, P.R. China
2
Received September 26, 2013; Accepted April 16, 2014 DOI: 10.3892/ol.2014.2121 Abstract. Sansalvamide A, a cyclic depsipeptide isolated from a marine fungus of the Fusarium genus, exhibits significant antitumor activity. In the present study, H‑10 (molecular formula, C38H55N5O6; molecular weight, 677.8732), a novel sansalvamide A derivative, demonstrated an inhibitory effect on the proliferation of murine melanoma B16 cells. It was confirmed that H‑10 induced the apoptosis of the B16 cells. The inhibitory rate of various concentrations of H‑10 on the B16 cells was measured by sulforhodamine B colorimetric assay, and the results revealed that the inhibitory effect of H‑10 on the B16 cells occurred in a concentration‑dependent manner. In addition, a growth curve model of the B16 cells treated with 50 µM H‑10 revealed that the effect of H‑10 also occurred in a time‑dependent manner. The apoptotic morphology of the B16 cells was observed using an optical microscope. Following the treatment of the cells with 50 µM H‑10 for 24 h, the cell apoptosis rate was analyzed using flow cytometry. The expression levels of caspase‑3, ‑8 and ‑9 were analyzed by western blot analysis, and the results indicated that H‑10 may induce the apoptosis of B16 cells. Introduction Malignant melanoma is a cancer with an increasing incidence and mortality rate. Furthermore, it is not sensitive to radiotherapy or chemotherapy, and thus presents a problem with regard to clinical treatment. Therefore, a number of studies have focused on the development of efficient and sensitive anticancer drugs. Sansalvamide A, a cyclic depsipeptide derived from a marine fungus (Fusarium), has been found to exhibit
Correspondence to: Professor Baoen Shan, Research Center, The
Fourth Hospital of Hebei Medical University, 12 Jiankang Road, Shijiazhuang, Hebei 050011, P.R. China E‑mail: [email protected]
Key words: sansalvamide A, B16, apoptosis, caspase‑3
significant antiproliferative effects in the National Cancer Institute's 60 cancer cell line panel (1,2). In recent years, the synthesis of sansalvamide A derivatives have received increasing attention. Novel sansalvamide A derivatives show improved anticancer abilities (3), suggesting that these novel compounds may prove to be valuable therapeutic agents. In the present study, a novel sansalvamide A derivative, H‑10, a cyclic pentapeptide (molecular formula, C38H55N5O6; molecular weight, 677.8732; Fig. 1), was investigated. Furthermore, this study focused on the effects of H‑10 on the growth and apotosis of rat malignant melanoma B16 cells. The results may provide a basis for future sutides of this novel compound. Materials and methods Materials. RPMI 1640, trypsin‑EDTA solution and fetal bovine serum (FBS) were purchased from Gibco‑BRL (Carlsbad, CA, USA). H‑10 cells were provided by the Hebei Province Key Laboratory of Molecular Chemistry for Drug (Shijiazhuang, China). Sulforhodamine B (SRB) was purchased from Tokyo Chemical Industry Co., Ltd (Tokoyo, Japan), and the bicinchoninic acid (BCA) kit was purchased from Shanghai Generay Biotechnology Co., Ltd. (Shanghai, China). The polyvinylidene fluoride (PVDF) membranes were purchased from Shanghai Generay Biotech Co., Ltd. The antibody against GAPDH (polyclonal rabbit anti-mouse) was purchased from Hangzhou Goodhere Biotechnology Co., Ltd. (Hangzhou, China). The antibodies against caspase-8, -9 and -3 (all polyclonal rabbit anti-mouse) were purchased from Bioworld Technology, Inc. (Minneapolis, MN, USA). The secondary fluorescence antibody (polyclonal goat anti-rabbit) was purchased from Nanjing Gene Biotech Co., Ltd. (Nanjing, China) The B16 cell line was stored at the Research Center of the Fourth Hospital of Hebei Medical University (Shijiazhuang, China). Cell culture. The cells were cultured in RPMI 1640 medium with 10% heat‑inactivated FBS and 100 µg/ml penicillin and streptomycin. The cell line was grown in 25‑cm 2 flasks in a humidified atmosphere of 5% CO2 at 37˚C, and the media were changed every second or third day. At 80‑90% confluence, the cells were digested with trypsin‑EDTA and plated in 25‑cm2
ZHANG et al: A NOVEL CYCLIC PENTAPEPTIDE, H‑10, INHIBITS B16 CELL PROLIFERATION
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Figure 1. Structure of sansalvamide A and its derivative, H‑10.
flasks with media changes every second or third day, on 24‑ or 96‑well plates.
control group was prepared simultaneously and a growth curve was generated.
Concentration‑dependent effect of H‑10 on B16 cell growth inhibition. H‑10 was dissolved in dimethyl sulfoxide (DMSO) and diluted with serum‑free medium to prepare solutions of 1,000, 500, 100, 10 and 1 µM. Single cell suspensions of B16 cells were prepared and adjusted to the indicated concentration. The cells were then inoculated in 96‑well plates (90 µl per well), with ~2,000 cells/well. Following 4 h of cell adherence, 10 µl H‑10 was added to each well to form final concentrations of 100, 50, 10, 1 and 0.1 µM. Each group was placed into three wells, while a 1% DMSO group was simultaneously prepared as the control. The SRB colorimetric method was used to calculate the percentage growth of the B16 cells treated with the various concentrations of H‑10 for 48 h.
Flow cytometric analysis of apoptotic cell death. At 80‑90% confluence, the cells were treated with 50 µM H‑10 for 24 h, while a control group was prepared. The treated and untreated cells were then harvested, washed with phosphate‑buffered saline (PBS) and fixed with 70% ethanol for 24 h. Next, the cells were centrifuged at 300 x g for 5 min and the pellet was resuspended in PBS containing 50 µg/ml propidium iodide and 10 µg/ml RNase A. The cells with 6 h. Finally, 100 µl Tris base was added and cells were agitated for 5 min. The optical density was recorded at a wavelength of 490 nm using a microplate reader. Time‑dependent effect of H‑10 on B16 cell growth inhibition. At 80‑90% confluence, the cells were harvested with trypsin, and serum‑free medium was used to produce a single‑cell suspension. The cells were then seeded in 24‑well plates at the concentration of 20,000 cells/well. After 24 h, the wells were replaced with fresh medium, including FBS. Next, the wells were treated with 50 µM H‑10 and the cell numbers were counted following 24, 48, 72, 96, 120 and 144 h. A
Detection of caspase‑8, ‑9 and ‑3 expression by western blot analysis. The cells at 80‑90% confluence were treated with 10, 30 and 50 µM H‑10 for 24 h, and a control group was prepared. The cellular protein was extracted by radioimmunoprecipitation assay lysis buffer and the concentration was measured using the BCA kit. A total of 50 µg protein was electrophoretically separated on a 10% polyacrylamide gel. The proteins were then transferred to a PVDF membrane under the conditions of 90 V and 200 mA for 60 min. Next, the membranes were incubated in antibody dilution solution (rabbit anti‑mouse caspase‑3, ‑8, ‑9 and GAPDH; 1:500) overnight at 4˚C. The blots were then incubated with the secondary fluorescence antibody (1:5,000) for 2 h in the dark, and the results were detected using the Odyssey infrared imager (LI‑COR, Inc., Lincoln, NE, USA). Statistical analysis. Statistical analysis was performed using SPSS version 13.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± standard error of the mean and were analyzed by t‑test. P0.05). Following the treatment of the B16 cells with the various concentrations of H‑10 (0.1, 1, 10, 50 and 100 µM) for 48 h, the proliferation rate of the B16 cells was found to gradually decrease. Furthermore, compared with control
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Figure 2. Effect of H‑10 on the proliferation of B16 cells measured using the sulforhodamine B colorimetric method. Compared with the control group, no significant difference was identified in the proliferation rate of the 1% DMSO group (P>0.05). H‑10 was found to cause concentration‑dependent inhibition of the B16 cells. **P
